The nuclear hormone receptor E75A regulates vitellogenin gene (Al‐Vg) expression in the mirid bug Apolygus lucorum

Apolygus lucorum is the predominant pest of Bacillus thuringiensis (Bt) cotton in China. 20‐hydroxyecdysone (20E) plays a key role in the reproduction of this insect. To better understand the mechanism underlying 20E‐regulated reproduction, the nuclear hormone receptor E75 isoform‐A of Ap. lucorum (Al‐E75A) was cloned and its expression analysed. A 2241‐bp sequence of Al‐E75A cDNA encoded an open reading frame of a polypeptide with a predicted molecular mass of 69.04 kDa. Al‐E75A mRNA was detected in female adult stages of Ap. lucorum with peak expression in 7‐day‐old animals. Al‐E75A was also expressed in several tissues, particularly in the fat body and ovary. A 3.2 kb Al‐E75A mRNA was detected in all tissues by Northern blot. The fecundity and longevity were significantly decreased in female adults treated with Al‐E75A small interfering RNA. The rates of egg incubation rates were considerably lower in the RNA interference‐treated animals compared to the untreated controls. In order to investigate the molecular mechanism underlying the effects described above, vitellogenin (Al‐Vg) was selected for further investigation. The expression pattern of Al‐Vg was similar to that of Al‐E75A and was up‐regulated by 20E. After knockdown of Al‐E75A, the expression profile of Al‐Vg and the protein levels were down‐regulated. These findings suggest that Al‐E75A plays a crucial role in the regulation of Al‐Vg expression in Ap. lucorum.     

Mushroom body‐preferential expression of proteins/genes involved in endoplasmic reticulum Ca2+‐transport in the worker honeybee (Apis mellifera L.) brain

To identify the molecular characteristics specific to the mushroom body (MB, a higher processing centre) neurones in the honeybee brain, we previously used proteomics to identify proteins that are preferentially expressed in these MBs. Here we continued our proteomic analysis to show that reticulocalbin, which is involved in endoplasmic reticulum (ER) Ca²⁺ transport, is also preferentially expressed in the MBs in the honeybee brain. Gene expression analysis revealed that reticulocalbin is preferentially expressed in the large‐type Kenyon cells, which are MB‐intrinsic neurones. In addition, the gene for the ryanodine receptor, which is also involved in ER Ca²⁺ transport, was also preferentially expressed in the large‐type Kenyon cells. In contrast, the expression of three other ER‐related genes, protein disulphide isomerase, sec61 and erp60, was not enriched in the MBs. These findings further support the notion that the function of ER Ca²⁺‐signalling, but not the mere intracellular density of ER, is specifically enhanced in the large‐type Kenyon cells in the honeybee brain.     

Silencing of Apis mellifera dorsal genes reveals their role in expression of the antimicrobial peptide defensin‐1

Like all other insects, two key signalling pathways [Toll and immune deficiency (Imd)] regulate the induction of honey bee immune effectors that target microbial pathogens. Amongst these effectors are antimicrobial peptides (AMPs) that are presumed to be produced by the nuclear factors kappa B (NF‐κB) Dorsal and Relish from the Toll and Imd pathways, respectively. Using in silico analysis, we previously proposed that the honey bee AMP defensin‐1 was regulated by the Toll pathway, whereas hymenoptaecin was regulated by Imd and abaecin by both the Toll and Imd pathways. Here we use an RNA interference (RNAi) assay to determine the role of Dorsal in regulating abaecin and defensin‐1. Honey bees have two dorsal genes (dorsal‐1 and dorsal‐2) and two splicing isoforms of dorsal‐1 (dorsal‐1A and dorsal‐1B). Accordingly, we used both single and multiple (double or triple) isoform knockdown strategies to clarify the roles of dorsal proteins and their isoforms. Down‐regulation of defensin‐1 was observed for dorsal‐1A and dorsal‐2 knockdowns, but abaecin expression was not affected by dorsal RNAi. We conclude that defensin‐1 is regulated by Dorsal (Toll pathway).     

Dissecting the role of Kr-h1 brain gene expression in foraging behavior in honey bees (Apis mellifera)

Expression of Krüppel homolog-1 (Kr-h1) in the honey bee brain is strongly associated with foraging behavior. We performed a series of studies to determine if Kr-h1 expression correlates with specific aspects of foraging. We found that Kr-h1 expression is unaffected by flight experience in male bees. Expression was unaffected by behavioral reversion of workers from foraging to brood care, suggesting that expression is not associated with the active performance of foraging, but rather with stable physiological changes. Kr-h1 expression is increased by cGMP treatment in workers, and the Kr-h1 promoter contains a conserved potential cGMP response element. Since cGMP treatment causes precocious foraging, our results suggest that Kr-h1 expression is associated with cGMP-mediated changes in the brain that occur early in the transition to foraging behavior.     

20‐Hydroxyecdysone stimulates nuclear accumulation of BmNep1, a nuclear ribosome biogenesis‐related protein in the silkworm,Bombyx mori

The pathway of communication between endocrine hormones and ribosome biogenesis critical for physiological adaptation is largely unknown. Nucleolar essential protein 1 (Nep1) is an essential gene for ribosome biogenesis and is functionally conserved in many in vertebrate and invertebrate species. In this study, we cloned Bombyx mori Nep1 (BmNep1) due to its high expression in silk glands of silkworms on day 3 of the fifth instar. We found that BmNep1 mRNA and protein levels were upregulated in silk glands during fourth‐instar ecdysis and larval–pupal metamorphosis. By immunoprecipitation with the anti‐BmNep1 antibody and liquid chromatography‐tandem mass spectrometry analyses, it was shown that BmNep1 probably interacts with proteins related to ribosome structure formation. Immunohistochemistry, biochemical fractionation and immunocytochemistry revealed that BmNep1 is localized to the nuclei in Bombyx cells. Using BmN cells originally derived from ovaries, we demonstrated that 20‐hydroxyecdysone (20E) induced BmNep1 expression and stimulated nuclear accumulation of BmNep1. Under physiological conditions, BmNep1 was also upregulated in ovaries during larval–pupal metamorphosis. Overall, our results indicate that the endocrine hormone 20E facilitates nuclear accumulation of BmNep1, which is involved in nuclear ribosome biogenesis in Bombyx.     

Vitellogenin expression in the ovaries of adult honeybee workers provides insights into the evolution of reproductive and social traits

Social insects are notable for having two female castes that exhibit extreme differences in their reproductive capacity. The molecular basis of these differences is largely unknown. Vitellogenin (Vg) is a powerful antioxidant and insulin-signalling regulator used in oocyte development. Here we investigate how Royal Jelly (the major food of honeybee queens) and queen mandibular pheromone (a major regulator of worker fertility), affect the longevity and reproductive status of honey bee workers, the expression of Vg, its receptor VgR and associated regulatory proteins. We find that Vg is expressed in the ovaries of workers and that workers fed a queen diet of Royal Jelly have increased Vg expression in the ovaries. Surprisingly, we find that expression of Vg is not associated with ovary activation in workers, suggesting that this gene has potentially acquired non-reproductive functions. Therefore, Vg expression in the ovaries of honeybee workers provides further support for the Ovarian Ground Plan Hypothesis, which argues that genes implicated in the regulation of reproduction have been co-opted to regulate behavioural differences between queens and workers.     

Identification of an E‐box DNA binding protein,activated protein 4, and its function in regulating the expression of the gene encoding diapause hormone and pheromone biosynthesis‐activating neuropeptide in Helicoverpa armigera

Activated protein 4 (AP‐4), an E‐box DNA‐binding protein, was cloned from the cotton bollworm, Helicoverpa armigera (Har). The expression of Har‐AP‐4 mRNA and the protein that it encodes are significantly higher in nondiapause pupae than in diapause pupae. In vitro‐translated Har‐AP‐4 can bind specifically to the E‐box motif on the promoter of the diapause hormone and pheromone biosynthesis‐activating neuropeptide (DH‐PBAN). Har‐AP‐4, fused with the green fluorescent protein (GFP), is localized to the nucleus, and overexpression of Har‐AP‐4 can significantly activate the promoter of the DH‐PBAN gene that is involved in nondiapause pupal development in H. armigera. These results suggest that Har‐AP‐4, which binds to the promoter of DH‐PBAN, may play a role in regulating pupal development in H. armigera.     

Hypoxic preconditioning of myoblasts implanted in a tissue engineering chamber significantly increases local angiogenesis via upregulation of myoblast vascular endothelial growth factor‐A expression and downregulation of miRNA‐1, miRNA‐206 and angiopoietin‐1

Vascularization is a major hurdle for growing three‐dimensional tissue engineered constructs. This study investigated the mechanisms involved in hypoxic preconditioning of primary rat myoblasts in vitro and their influence on local angiogenesis postimplantation. Primary rat myoblast cultures were exposed to 90 min hypoxia at <1% oxygen followed by normoxia for 24 h. Real time (RT) polymerase chain reaction evaluation indicated that 90 min hypoxia resulted in significant downregulation of miR‐1 and miR‐206 (p < 0.05) and angiopoietin‐1 (p < 0.05) with upregulation of vascular endothelial growth factor‐A (VEGF‐A; p < 0.05). The miR‐1 and angiopoietin‐1 responses remained significantly downregulated after a 24 h rest phase. In addition, direct inhibition of miR‐206 in L6 myoblasts caused a significant increase in VEGF‐A expression (p < 0.05), further establishing that changes in VEGF‐A expression are influenced by miR‐206. Of the myogenic genes examined, MyoD was significantly upregulated, only after 24 h rest (p < 0.05). Preconditioned or control myoblasts were implanted with Matrigel? into isolated bilateral tissue engineering chambers incorporating a flow‐through epigastric vascular pedicle in severe combined immunodeficiency mice and the chamber tissue harvested 14 days later. Chambers implanted with preconditioned myoblasts had a significantly increased percentage volume of blood vessels (p = 0.0325) compared with chambers implanted with control myoblasts. Hypoxic preconditioned myoblasts promote vascularization of constructs via VEGF upregulation and downregulation of angiopoietin‐1, miR‐1 and miR‐206. The relatively simple strategy of hypoxic preconditioning of implanted cells ‐ including non‐stem cell types – has broad, future applications in tissue engineering of skeletal muscle and other tissues, as a technique to significantly increase implant site angiogenesis.     

Division of labour in honey bees: age‐ and task‐related changes in the expression of octopamine receptor genes

The honey bee (Apis mellifera L.) has developed into an important ethological model organism for social behaviour and behavioural plasticity. Bees perform a complex age‐dependent division of labour with the most pronounced behavioural differences occurring between in‐hive bees and foragers. Whereas nurse bees, for example, stay inside the hive and provide the larvae with food, foragers leave the hive to collect pollen and nectar for the entire colony. The biogenic amine octopamine appears to play a major role in division of labour but the molecular mechanisms involved are unknown. We here investigated the role of two characterized octopamine receptors in honey bee division of labour. AmOctαR1 codes for a Ca²⁺‐linked octopamine receptor. AmOctβR3/4 codes for a cyclic adenosine monophosphate‐coupled octopamine receptor. Messenger RNA expression of AmOctαR1 in different brain neuropils correlates with social task, whereas expression of AmOctβR3/4 changes with age rather than with social role per se. Our results for the first time link the regulatory role of octopamine in division of labour to specific receptors and brain regions. They are an important step forward in our understanding of complex behavioural organization in social groups.     

Optogenetic control of iPS cell‐derived neurons in 2D and 3D culture systems using channelrhodopsin‐2 expression driven by the synapsin‐1 and calcium‐calmodulin kinase II promoters

Development of an optogenetically controllable human neural network model in three‐dimensional (3D) cultures can provide an investigative system that is more physiologically relevant and better able to mimic aspects of human brain function. Light‐sensitive neurons were generated by transducing channelrhodopsin‐2 (ChR2) into human induced pluripotent stem cell (hiPSC) derived neural progenitor cells (Axol) using lentiviruses and cell‐type specific promoters. A mixed population of human iPSC‐derived cortical neurons, astrocytes and progenitor cells were obtained (Axol‐ChR2) upon neural differentiation. Pan‐neuronal promoter synapsin‐1 (SYN1) and excitatory neuron‐specific promoter calcium‐calmodulin kinase II (CaMKII) were used to drive reporter gene expression in order to assess the differentiation status of the targeted cells. Expression of ChR2 and characterisation of subpopulations in differentiated Axol‐ChR2 cells were evaluated using flow cytometry and immunofluorescent staining. These cells were transferred from 2D culture to 3D alginate hydrogel functionalised with arginine‐glycine‐aspartate (RGD) and small molecules (Y‐27632). Improved RGD‐alginate hydrogel was physically characterised and assessed for cell viability to serve as a generic 3D culture system for human pluripotent stem cells (hPSCs) and neuronal cells. Prior to cell encapsulation, neural network activities of Axol‐ChR2 cells and primary neurons were investigated using calcium imaging. Results demonstrate that functional activities were successfully achieved through expression of ChR2‐ by both the CaMKII and SYN1 promoters. The RGD‐alginate hydrogel system supports the growth of differentiated Axol‐ChR2 cells whilst allowing detection of ChR2 expression upon light stimulation. This allows precise and non‐invasive control of human neural networks in 3D.     

Impact of polymorphisms of the major histocompatibility complex class II,interleukin‐10, tumor necrosis factor‐α and cytotoxic T‐lymphocyte antigen‐4 genes on inhibitor development in severe hemophilia A

Summary. Background: Approximately 25% of severe hemophilia A (HA) patients develop antibodies to factor VIII protein. Patients: In the present case‐controlled cohort study, 260 severely affected, mutation‐type‐matched HA patients were studied for association of human leukocyte antigen (HLA) class II molecules and polymorphisms in the genes encoding interleukin‐10 (IL‐10), tumor necrosis factor‐α (TNF‐α) and cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4) and development of inhibitors. Results: Our results demonstrate a higher frequency of DRB1*15 and DQB1*0602 alleles as well as of the haplotype DRB1*15/DQB1*0602 in inhibitor patients [odds ratio (OR) 1.9; P < 0.05]. In TNF‐α, the A allele of the ?308G>A polymorphism was found with higher frequency in the inhibitor cohort (0.22 vs. 0.13, OR 1.80). This finding was more pronounced for the homozygous A/A genotype (OR 4.7). For IL‐10, the ?1082G allele was observed more frequently in patients with inhibitors (0.55 vs. 0.43; P = 0.008). The functional cytokine phenotype was determined for the first time, on the basis of the genetic background, and this showed that 12% of patients with inhibitors were high‐TNF‐α/high‐IL‐10 producers, as compared with 3% of non‐inhibitor patients (OR 4.4). A trend for a lower frequency of the A allele of the CT60 polymorphism in CTLA‐4 was found in inhibitor patients (0.42 vs. 0.50). Conclusions: In conclusion, the reported data clearly highlighted the participation of HLA molecules in inhibitor formation in a large cohort of patients. The higher frequencies of the ?308G>A polymorphism in TNF‐α and ?1082A>G in IL‐10 in inhibitor patients confirmed the earlier published data. The CT60 single‐nucleotide polymorphism in CTLA‐4 is of apparently less importance.     

Rationale and design of the CANARI study: a case–control study investigating the association between prostate cancer and 5‐alpha‐reductase inhibitors for symptomatic benign prostate hypertrophy by linking SNIIRAM and pathology laboratories in a specific region in France

Benign prostate hypertrophy (BPH) could be associated with low urinary symptoms requiring medical treatment: 5‐alpha‐reductase inhibitors (5‐ARI) or ɑ‐blockers. Two clinical trials investigating 5‐ARI use in prostate cancer (PCa) primary prevention highlighted a potential safety signal with an increased risk of high‐grade PCa. Later observational studies failed to show similar results but have some limits. This paper focuses on describing the protocol of the CANARI study and its feasibility, as regards the matching process of two pseudo‐anonymous databases. The study concerned patients living in the Brittany region (France) between 2010 and 2013. We designed a case–control study nested within a cohort of men treated by medical drugs licensed for symptomatic BPH between 2010 and 2011. Cases were patients with incident PCa diagnosed between 2012 and 2013 identified through French Health database (SNIIRAM). Gleason score was searched through Brittany pathology laboratories. Controls were patients without PCa diagnosis. Local pathology laboratories database was constituted in Brittany, gathering Gleason scores. No unique identification number is available in France; linkage of SNIIRAM and Brittany pathology laboratories database was made by deterministic matching. We matched 859 cases to Gleason grading (119 had Gleason score ≥8 and 740 had Gleason <8); around 22% of cases received 5‐ARI and 78% α‐blockers or phytotherapy. The CANARI study investigated in a population of men treated for BPH the risk of PCa with 5‐ARI, according to Gleason grade thanks to SNIIRAM database enriched by local pathological results.